302 INFECTION AND IMMUNITY 



if, after coagulation, the clot is separated from the glass with a 

 sterile platinum wire. 



In obtaining blood from larger animals, horses, sheep, etc., a 

 cannula may be introduced into the jugular or internal saphenous 

 veins. The skin is shaved and sterilized and a rubber tourniquet 

 placed about the neck or thigh, as the case may be, in order to 

 cause the vein to stand out. A small incision may be made through 

 the skin over the vein, but is not necessary. A cannula, with rubber 

 tubing attached, is then plunged into the vein and the blood caught 

 in sterile high cylindrical jars, allowed to clot, and placed in the 

 refrigerator. The serum is taken off . after twenty-four to forty- 

 eight hours with sterile pipettes. 



Agglutination Tests. For the determination of the agglutinating 

 power of serum it is necessary to make suitable dilutions of the 

 serum, and to prepare an even emulsion of the microorganisms to 

 be tested. The test may be made microscopically or macroscopically. 

 The microscopic test is the one in general use in the diagnosis of 

 typhoid fever, and is occasionally applied to some other diseases. 

 In its application to typhoid fever it is usually spoken of as the 

 Gruber-Widal reaction. 



Twelve- to eighteen-hour broth cultures of the typhoid bacillus, grown 

 at incubator temperature, may be used. It is preferable, however, to use 

 an emulsion of a twelve to twenty-four hour old ag'ar culture in physiological 

 salt solution (0.85 per cent). The salt-solution emulsion is made by adding 

 about 10 c.c. of normal salt solution to the fresh agar slant culture, carefully 

 detaching the culture from the surface of the agar with a flexible platinum 

 wire, and pipetting off the emulsion thus made. With some microorganisms 

 it is sufficient simply to allow the larger clumps to settle and to pipette 

 off the supernatant turbid emulsion. With other microorganisms, the tendency 

 to form clumps makes it necessary to resort to further methods of securing 

 an even distribution of the bacteria. This may be done either by sucking 

 the emulsion in and out through a narrow pipette held perpendicularly against 

 the bottom of a watch glass, as in Wright's technique for the opsonic test 

 (see section on opsonins, p. 339), or by carefully rubbing the clumps against 

 the watch glass with a stiff platinum wire. In the case of the tubercle 

 bacillus not even this suffices, but it becomes necessary to grind the moist 

 bacillary masses in a mortar before emulsifying. With the tubercle bacillus, 

 too, it is preferable to use salt solution at 1.5 per cent concentration. 



The serum dilutions are obtained by first making a one to ten 

 dilution of serum with normal salt solution. The serum used for 



